Nuclear magnetic resonance (NMR) spectroscopy is an analytical method that provides a wealth of chemical and structural information from complex biological samples. Unfortunately, NMR is an inherently insensitive technique which requires larger sample amounts than other chemical characterization methods. The overall goal of this proposal is to continue nanoliter-volume NMR probe development so that 5 - 1000 nanoliter volumes and picomole masses can be analyzed. The fundamental advance that enables such sensitivity' improvements is miniaturized radio- frequency coils for NMR signal detection. A major portion of this work involves the optimization of coil geometry and fabrication to maximize sensitivity and minimize spectral linewidth, to add a broad range of heteronuclear NMR capabilities, and to develop multiple microcoil probes. Probes with up to 16 microcoils will dramatically improve NMR throughput. The combination of NMR and separation methods provides unmatched structural elucidation capabilities. Specifically, optimized flow cells, acquisition parameters, separation modes and preconcentration methods will be developed for capillary electrophoresis and capillary liquid chromatography. A unique series of on-line NMR techniques will be developed to monitor the separation process including on-line temperature, flow rate and imaging techniques. The implementation of this technology greatly expands biological applications where mass limitations currently prevent NMR structural determinations. Specifically, diffusion-ordered NMR will be used to obtain quantitative information on the diffusion rates in specific populations of cellular organelles from a series of model cells from the marine mollusk Aplysia californica. Secondly, single cell NMR spectroscopy will be developed for identified neurons in Aplysia californica which will allow the major osmolytes and cytoplasmic and nuclear components to be measured in intact neurons, as well as the physico-chemical environment of these compartments. Thus, the nanoliter- volume NMR probes developed during this research will offer significantly improved mass sensitivity for a widening range of NMR analysis, enable NMR to be used with a variety of microseparation methods and allow a new range of biological applications.
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